Hyaluronan (HA) esterification via acylation technique for moldable devices

ABSTRACT

A series of novel, melt- or mold-processable HA esters with varying aliphatic chain lengths are synthesized from silyl HA-quaternary (quat.) ammonium salt complex (preferably silyl HA-CTA, a silylated HA complex with cetyltrimethyl ammonium salt). Introduction of aliphatic acyl groups, preferably acid chlorides, to disrupt the strong HA intermolecular bonding, is done via acylation. Acylation takes place at the oxygen of the trimethylsilyloxy group —O—Si(CH 3 ) 3  in the silyl HA-CTA by removal of trimethylsilyl groups therefrom. Optionally, crosslinking may be performed during the shaping/molding of the HA esters into a structure/device, or thereafter, if at all. Native HA can then be regenerated/recovered by saponification hydrolysis, removing acyl groups, —CH 3 (CH 2 ) 10 CO, and the cetyltrimethyl ammonium salt groups, -CTA, from HA ester. The structure/device of a preselected shape (e.g., porous or solid, bulk structure or fibers) may become a component of an assembly, a product that is further processed, integrated into another component (e.g., laminated, adhered, assembled, further shaped, chemically-intermixed/intermingled), and so on.

BACKGROUND OF THE INVENTION

In general, the present invention relates to the synthesis and use ofhyaluronan (a.k.a., hyaluronic acid, sodium hyaluronate, or HA), andother hydrophilic polymers with pendant hydroxy groups that are notgenerally melt-processable in their ‘native’ state. As a naturallyoccurring polysaccharide with a large unbranched structure consisting ofrepeating disaccharides of N-acetylglucosamine and glucuronic acid, thestructure of shown in FIG. 2A, present in vertebrate tissues and bodyfluids, HA has certain physical and biological properties, includingviscoelasticity, hydrophilicity, lubricity and cell-activity regulation.Native HA and its currently available derivatives degrade beforemelting; thus, they cannot be thermally molded into custom shapes orotherwise thermally integrated with thermoplastic biomaterials. Inbiomechanical applications where characteristics such as maintaining ashape and/or supporting or protecting/cushioning a structure (e.g.,joints, bone, cartilage and other tissue) are important, as well as forsurgery instruments and aids, etc. and other mechanisms (whetherbiocompatibility is an issue)—degradation prior to melt point is anobstacle to using HA. The extreme hydrophilicity of HA does not permituse in conjunction with durable, hydrophobic biomaterials, such aspolyethylene or polypropylene. Furthermore, the quick turnover within ananimal body limits use of native HA in applications such as longer-termand permanent implants.

More-specifically, the instant invention is directed to a novel melt- ormold-processable hydrophilic polyanionic polymer with pendant hydroxygroups that are not generally melt processable in their native state,such as HA, as well as a method of synthesis of such a polymer. Ofparticular interest is hyaluronan/hyaluronic acid/sodium hyaluronate(generally, throughout referred to as “HA”); produced according to theinvention, the melt- or mold-processable HA has a melting point belowthe point at which the polymer degrades. Pure (whether synthetic orgenetically engineered, or native) HA has a melting point above thepoint of substantial degradation making it by-and-large impossible tomold or shape into structures suitable for use. A polymeric materialproduced according to the unique technique of the invention, provides aprocessable HA polymer that, once re-hardened (i.e., cooled or otherwisesolidified), can be used in a variety of product applications, whetherresultant structures are used ‘as is’ having been molded, extruded, orotherwise shaped into a mechanism/piece/device/etc. or employed as amember, component or subassembly of an assembly/system.

According to the invention a series of novel, melt- or mold-processableHA esters with varying aliphatic chain lengths are synthesized fromsilyl HA-quat. ammonium salt complex (preferably silyl HA-CTA, asilylated HA complex with cetyltrimethyl ammonium salt, a hyaluronanderivative). Introduction of aliphatic acyl groups (e.g., acid chlorideslisted, TABLE 1) to HA disrupts the strong HA intermolecular bonding,reducing the crystallinity and producing appreciablethermoplasticization. Acylation takes place at the oxygen of thetrimethylsilyloxy group —O—Si(CH₃)₃ in the silyl HA-CTA by removal oftrimethylsilyl groups therefrom. Optionally, crosslinking may beperformed during the shaping/molding of the HA esters into astructure/device, or thereafter, if at all. Native HA can then beregenerated/recovered by saponification/hydrolysis, removing acyl and-CTA groups. The structure/device of a preselected shape (e.g., porousor solid, bulk structure or fibers, etc.) may become a component of anassembly, a product that is further processed (e.g., seeded with cells,further shaped, etc.), integrated into another component (e.g.,laminated, adhered, assembled, further shaped/molded together with acomponent, chemically-intermixed/intermingled, etc.), and so on.

Structure(s) produced according to the invention may be composedentirely of the melt- or mold-processable derivatized HA (or otherpolymer with hydroxy groups) and used alone, or used as scaffold forbiological materials (e.g., cells, morphogenic proteins) or incorporatedinto a component, piece, module, feature, or mechanism/structure/memberto produce a ‘system’ such that the HA's hydrophilic outer surfaceprovided is interior- or exterior-facing, etc. A non-exhaustive list ofpossibilities contemplated for use of the derivative HA of theinvention—including those where a generally hydrophilic outer surface isdesirable—include: bearing surfaces or components for items such asgears, fishing rod eyelets, bearings of all types, joint and otherweight-bearing mechanisms, whether incorporated as part of manufacturingequipment, as part of the manufactured product itself, etc.; flexiblebarrier surfaces separating a first and second area (such flexiblebarriers to include the membrane material or tubing used for catheterballoons, catheter tubing, hot air balloons, condoms, IV tubing,diaphragms, flexible bladders, etc.); transparent member surfacesincluding the transparent planar or curved polymeric films and sheetmaterial used where optical clarity is sought, such as for fish tanks,polymeric covers for vehicle, water- or aircraft head-lamps andblinkers/fog-lights, covers for spot-lights, windows on or in a vehicle,aircraft, watercraft, and spacecraft, monitor and television screens,ophthalmic lenses, camera lenses and view-finders, etc.; in vivoimplants of any of a variety of total or partial joint replacements,splints, stents, diaphragms, etc.; drag reduction surfaces andassociated components of a vehicle, watercraft, aircraft and spacecraftsuch as hulls, pontoons, vehicle-body parts, blades/runners, etc., aswell as the glide-surface of snowboards, water and snow skis, etc.;reaction resins for research or industrial components; topical dressingsurfaces for dressings such as those used for medical/veterinaryapplications such as adhesive bandages, sterile pads for wounds andsurgical procedures, bandage tape/adhesive, ace bandages, soft casts,etc.; and dental splints (to include mouth-guards, tooth/jaw-correctionsplints, etc.).

Further examples of applications for the invention include: tissueengineered scaffolds (porous, seeded with cells, etc.) for cartilage andother tissue repair and treatment, wound dressings, artificial skin,viscoelastics for intra-surgical protection and prevention ofpost-operative adhesions, hydrophilic, lubricious and/or anti-foulingand/or anti-coagulant coatings (e.g., catheters, contact lenses,dialysis membranes), drug release/delivery devices, and biodegradeablematerials (e.g., nerve guides).

While the focus, here, is of HA (i.e., a hydrophilic polyanionic polymerwith hydroxy groups that is not generally melt processable in currentlyavailable native or derivative state(s)) other biomaterials exhibitingsimilar characteristics that would benefit from molding or shaping arecontemplated hereby as a starting polymer. Examples shown hereinshowcase the synthesis of unique HA esters, i.e., melt- ormold-processable HA derivatives having hydrophobicity and compatibilitywith other generally hydrophobic materials. Derivatization of HAaccording to the invention permits control of its hydrophobicity,expanding the range of useful solvents and non-solvents during synthesisand fabrication into end product. In the spirit and scope of designgoals contemplated hereby, the novel melt- or mold-processed polymerproduced according to the invention may be made by chemically modifyinga wide variety of hydrophilic polyanionic polymers containing pendanthydroxy groups, including without limitation: polyanionic polyhydroxypolymers such as polysaccharides and glycoaminoglycans.

Hyaluronan was first isolated from bovine vitreous humor in acid form in1934; it was coined “hyaluronic acid” meaning uronic acid from hyaloid(vitreous). The first non-inflammatory fraction of sodium hyaluronate,called NIF—NaHA, which was free of impurities that could causeinflammatory reactions was synthesized by Endre Balazs. HA has certainphysical and biological properties: (1) Viscoelasticity. Hyaluronancarries one carboxyl group (—COOH) per disaccharide unit, which isdissociated at physiological pH thereby conferring a polyanioniccharacteristic to the compound; (2) Hydrophilicity. Hyaluronan acts as awater-retaining polymer network in many tissues; it can hold largeamounts of water like a molecular sponge. The hydrodynamic volume of HAin solution is 1000 times larger than the space occupied by theunhydrated polysaccharide chain. (3) Lubricity. The extraordinaryviscoelastic properties also make hyaluronan ideal as a lubricant.Hyaluronan in synovial fluid complexes with proteins and penetrates thesurface of cartilage, forming a layer of HA protein complex that servesas a lubricating layer in joints and other tissue surfaces that slidealong each other. Under slow mechanical loading it behaves as a viscousoil-like lubricant. At higher mechanical loading rates the HA layerbecomes a highly deformable elastic system: it absorbs and converts animposed stress into an elastic deformation, then rebounds to theoriginal condition when the stress is removed.

Not only does HA act as a vital structural component of connectivetissues, it also plays an role in diverse biological processes, such ascellular migration, mitosis, inflammation, cancer, angiogenesis andfertilization. However, HA's high solubility, rapid degradation andshort residence time in water have historically limited biomedicalapplication of naturally occurring HA, particularly in tissueengineering and viscoseparation applications. Four pendant groups onhyaluronan are available (FIG. 2A) for chemical modification: carboxyl,hydroxyl, acetamido group and the reducing end-group. Prior attempts ofothers at synthesizing HA derivatives have been directed at modifyingone or more of these groups, resulting in much different derivativeend-product (none have thermoplastic characteristics, as theyby-and-large have no meltable endotherms).

Two groups of commercialized hyaluronan derivatives, include HYLAN andHYAFF®: HYLAN is a brand name for a crosslinked hyaluronan in whichcrosslinking only occurs on hydroxyl groups, not affecting carboxyl andacetamide groups, this cross-linking was done by Balazs and hiscolleagues. HYAFF® is the brand name of a class of hyaluronan esterswith the free carboxyl group of glucuronic acid esterified usingdifferent types of alcohols (delta Valle et al., U.S. Pat. No.4,851,521, 1989). patent application No. US 2002/0143171 A1 published 3Oct. 2002 to Yui et al. focuses on a chemically modified HA for use aspharmaceuticals, foodstuffs, cosmetics (e.g., moisturizing agent,lotion) and similar flowable, gel-like substances, but their crosslinkedpolymer can not melt without first degrading. Yui et al. modified thehydroxyl groups of HA with an acid halide carrying photoreactive groups,such as cinnamoyl chloride, in DMF solution in the presence of pyridine.The product was dissolved in DMF, and was subjected to ultravioletradiation to crosslink HA. The applicants hereof have published earlierwork: US App. US 2003/0083433 A1 filed on behalf of the assignee hereoffor the applicants on 29 Oct. 2002, and is hereby incorporated herein byreference to serve as technical background support.

‘Animal’ as used throughout includes any multicellular organism having abody that can move voluntarily and actively acquire food and digest itinternally, including human beings and other mammals, birds and fish.‘Extrude’/‘extrusion’ as used includes a molding technique as follows:the moldable material is forced through the shaping die of an extruder;may have a solid or hollow cross section. The following generalacronyms, if used throughout, are decoded on the following page:

-   HA—Hyaluronic acid/hyaluronan/sodium hyaluronate.-   HA-CPC—the complex of HA polyanion and cetylpyridinium salt.-   HA-CTA—the complex of HA polyanion and cetyltrimethyl ammonium salt.-   HA⁻-QN⁺—the complex of HA polyanion & long-chain paraffin ammonium    cation.-   HMDS—hexamethyldisilazane, a silylation agent.-   THF—Tetrahydrofuran.-   TMCS—trimethylchlorosilane, a silylation agent.-   QN⁺—long-chain paraffin ammonium cation.

SUMMARY OF THE INVENTION

It is a primary object of the invention to provide a process forproducing a hyaluronan (HA) ester having mold- or melt-processablecharacteristics. The HA ester is synthesized by (a) performing asilylation reaction on an HA-quaternary ammonium complex and (b)performing an acylation reaction on the silyl HA-quaternary ammoniumcomplex using an acid chloride. Also disclosed as featured herein, arethe associated unique HA ester and shape-structures made out of theunique HA ester and out of regenerated HA, as well as the HA ester asfurther processed and/or integrated into an assembly or system. While HAis the focus, also contemplated within the spirit and scope are otherhydrophilic polyanionic polymers containing pendant hydroxy groups arecontemplated as starting material—including without limitation,polyanionic polyhydroxy polymers such as polysaccharides andglycoaminoglycans.

There are numerous further distinguishing features of the process andresulting HA ester. The step of performing a silylation reactionpreferably includes silyating an HA-cetyltrimethyl ammonium saltcomplex, HA-CTA, which produces a silyl HA-cetyltrimethyl ammonium saltcomplex. The step of performing an acylation reaction preferablyincludes introducing an acid chloride such as one of the followingaliphatic acyl groups: Hexanoyl, CH₃(CH₂)₄COCl; Octanoyl, CH₃(CH₂)₆COCl;Decanoyl, CH₃(CH₂)₈COCl; Lauroyl, CH₃(CH₂)₁₀COCl; Palmitoyl,CH₃(CH₂)₁₄COCl; and Stearoyl, CH₃(CH₂)₁₆COCl. The hyaluronan (HA) estercan be shaped by applying thermal energy to melt-process the ester intoa structure-shape, or by applying pressure to shape the HA ester. A widevariety of structure-shapes are contemplated, including withoutlimitation: polymer fibers, generally solid bulk or porous bulkstructure—the bulk structure shape can be that of a thin layer or othergenerally planar shape, an irregular shape such as that for an implantor tissue, a classic regular shape such as cylindrical, torus, block,and so on. The porous structure can be ‘seeded’ with cells.

A saponification substantially removing acyl groups, —CH₃(CH₂)_(N)CO,and the cetyltrimethyl ammonium salt groups, -CTA, from the HA ester toproduce a regenerated HA, can be performed after an initial shaping theester (e.g., box 17 to box 34, FIG. 1), or prior to shaping into apreselected structure-shape (e.g., from box 16 via route 33 a to box34). The step of shaping the HA ester into a structure-shape can be donein conjunction with a crosslinking of the HA ester.

In another aspect, a hyaluronan (HA) ester is produced from an acylatedsilyl HA-cetyltrimethyl ammonium salt complex, wherein an acylationagent is used in producing the complex and comprises an acid chlorideselected from the group consisting of: Hexanoyl, CH₃(CH₂)₄COCl;Octanoyl, CH₃(CH₂)₆COCl; Decanoyl, CH₃(CH₂)₈COCl; Lauroyl,CH₃(CH₂)₁₀COCl; Palmitoyl, CH₃(CH₂)₁₄COCl; and Stearoyl, CH₃(CH₂)₁₆COCl.Once again, the HA ester can be shaped into a structure-shape that issaponified into a regenerated HA form, and the structure-shapeintegrated with a component. The HA ester preferably having beenproduced from an acylated silyl HA-cetyltrimethyl ammonium salt complex.

As can and will be appreciated, certain of the many unique features, aswell as the further-unique combinations thereof, supported andcontemplated hereby within the spirit and scope of this disclosure, mayprovide a variety of advantages. The advantages of the new features andcombinations disclosed hereby will be appreciated, especially byproviders of medical and veterinary care and services and products(where biocompatibility with animal tissue and functionality is aconsideration), by perusing the instant technical discussion, includingdrawings, claims, and abstract, in light of drawbacks to traditionaldevices and known materials identified throughout, or as may beuncovered.

The unique melt-processable HA material and process for producing,provide design options and versatility to accommodate a wide variety ofapplications. The basic novel structure and characteristics of themelt-processable HA material of the invention makes it adaptable for usein fabricating a wide variety of configurations/shapes (porous or solid,bulk structure or fibers, etc.) and sizes, with or without crosslinkingwith other material(s). The unique approach taken for producing themelt-processable HA material is reproducible and can be carried outwithout the need to employ reaction catalysts.

BRIEF DESCRTIPTION OF THE DRAWINGS

For purposes of illustrating the innovative nature plus the flexibilityof design and versatility of preferred and alternative mold- andmelt-processable. HA material and associated method of producing,supported and disclosed hereby, the invention will be better appreciatedby reviewing accompanying drawings (in which like numerals, if used,designate like or similar parts). One will appreciate the features thatdistinguish the instant invention from conventional structures andapplications. The drawings have been included to communicate features ofthe innovative core and further unique process of producing andassociated structure, the HA material of the invention, as well as todemonstrate the unique approach taken, by way of example only, and arein no way intended to unduly limit the disclosure hereof.

FIG. 1 is a flow diagram depicting details of a process 10 of producingthe polymeric HA derivative material and structure(s) according to theinvention—illustrated are core, as well as further distinguishingfeatures, resulting in chemical structures such as those represented, byway of example only, in FIGS. 2A-2C.

FIG. 2A depicts a chemical structure of ‘native’ hyaluronan (HA).

FIGS. 2B-2C depict the synthesis of HA esters according to the inventionwherein an acid chloride is used as acylation agent: FIG. 2B depictsalternative structures of HA with R=H as the HA⁻-QN⁺ complex, and as asilylated HA⁻-QN⁺ complex structure in which R=Si(CH₃)₃; and FIG. 2Cdepicts the structure of an acylated silylated HA⁻-QN⁺ complex whereinSi(CH₃)₃ groups are replaced by R′CO, with R′=CH₃(CH₂)_(n).

FIG. 3 is a graphical representation of Fourier Transform InfraredSpectroscopy (FT-IR) spectra of silyl HA-CTA, HA palmitate and HA-CTA.

FIG. 4A-4B are graphical representations of high-resolution XPS C1sspectra of binding energy as labeled: FIG. 4A is of HA laurate; FIG. 4Bis of silyl HA-CTA.

FIG. 5A-5B are graphical representations of high-resolution XPS N1sspectra of binding energy as labeled: FIG. 5A is of HA laurate; FIG. 5Bis of silyl HA-CTA.

FIG. 6 is a graphical representation of FT-IR spectra of regenerated HAfrom saponification (top curve as labeled) and ‘native’ HA (bottom curveas labeled).

FIG. 7 is a graphical representation of Thermal Gravimetric Analysis(TGA) done for HA, HA caproate, and HA laurate (curves as labeled).

FIG. 8 is a graphical representation of Differential ScanningCalorimetry (DSC) done for HA, HA caproate, and HA laurate (curves aslabeled).

DETAILED DESCRIPTION OF EMBODIMENTS IN DRAWINGS

Reference will be made back-and-forth to the several figures inconnection with discussion of the examples as well as the features ofthe invention: FIG. 1 details features of a method 10 for producing HAesters, regenerated HA, and structure-shapes of the invention in aflow-diagram format, to better appreciate the features of the HAstructures depicted in FIGS. 2A-2C.

The FIG. 1 flow diagram depicts details of a process 10 of producing themold- and melt-processable material and structure(s) according to theinvention—illustrated are core, as well as further distinguishingfeatures, resulting in chemical structures such as those represented, byway of example, in FIGS. 2A-2C. FIG. 2A depicts a chemical structure of‘native’—or, sometimes referred to as plain—hyaluronan (HA). FIGS. 2B-2Cdepict the synthesis of HA esters according to the invention wherein anacid chloride is used as acylation agent. FIG. 2B depicts alternativestructures of the hydrophilic polyanionic polymer HA, as shown in FIG.2B with R=H as an HA⁻QN⁺ complex (step 12, FIG. 1), and as a silylatedHA⁻-QN⁺ complex structure in which R=Si(CH₃)₃ (step 14, FIG. 1). FIG. 2Cdepicts the structure of an acylated silylated HA⁻-QN⁺ complex whereinSi(CH₃)₃ groups are replaced by R′CO, with R′=CH₃(CH₂)_(n) (box 16, FIG.1). As mentioned, and as further described in detail by way of example,the HA⁻-QN⁺ complex can be an HA-cetyltrimethyl ammonium salt complex,HA-CTA, whereby silylating produces a silyl HA-CTA complex.

As represented by FIG. 2C, the silylated complex of FIG. 2B has beenacylated according to the invention to produce the mold- ormelt-processable polymer (HA ester) after having performed step 16,FIG. 1. As one can appreciate (FIG. 2B), the cloaking of HA⁻-QN⁺complexes results in the hydrophilic groups being replace with silylatedfunctional groups: hydrogen (H) has been replace with Si(CH₃)₃; and oncean acylation of the FIG. 2B polymer complexes using an acid chloride(preferably one having a hydrocarbon tail of sufficient length such thatthe complexes have an opportunity to become mold- or melt-processable)has been performed (step 16, FIG. 1), an HA ester complex such as thatdepicted in FIG. 2C results. The example represented by FIG. 2B is asilylated HA⁻-QN⁺ complex that has been acylated wherein the Si(CH₃)₃groups are replaced by R′CO, with R′=CH₃(CH₂)_(n).

Returning to FIG. 1, a method 10 of producingstructure(s)/devices/product according to the invention isillustrated—as mentioned, included are core, as well as furtherdistinguishing, features of the invention. Steps of the method include:Produce complexes of a hydrophilic polymer such as HA (box/step 12).Temporarily cloak (e.g., preferably via silylation) at least a portionof the hydrophilic groups of the HA-quat. ammonium salt complex. Performesterification employing a suitable process such as acylation of thepolymer complexes using an acid chloride (box 16). As noted in FIG. 1(box 16) acylation takes place at the oxygen site of thetrimethylsilyloxy group —O—Si(CH₃)₃ in the silyl HA-CTA, astrimethylsilyl groups of silyl HA-CTA are more easily replaced thanactive hydrogen in the hydroxyl (—OH) groups of non-silylated HA⁻-QN⁺(i.e., the location of the Si(CH₃)₃ groups is where the acid chloride‘attacks’ the FIG. 2B silylated HA-CTA complex(es)).

A desired shape can then be attained by pressure and/orthermal/thermo-processing employing any of a number of suitable processwhereby the lower melt temperature may be attained and pressuresufficient to produce the end-structure(s)/device(s)/piece(s) (box/step17). A wide variety of structure-shapes are contemplated, as explainedherethroughout, which may be attained by employing a wide variety ofwell known molding/shaping processes. As in FIG. 1, optionally, acrosslinking of resultant polymeric structures may be performed atdifferent points within process 10: steps 31 and 36. Next, removal ofacyl groups and -CTA groups from the polymer complexes in solution maybe done; e.g., dissolving the HA ester in an organic solvent blend(e.g., NaCl for hydrolysis—the organic solvent is selected based on HAester structure) and using an aqueous alkaline solution to saponify, ascollectively identified as step 34. Thus producing a generallyhydrophilic outer surface on a structure-shape made out of the HA ester.

Flexibility of use of the novel HA ester synthesized according to theinvention can be appreciated (see FIG. 1). Further processing intoselected product structure-shape may be done as shown, by way ofexample, in FIG. 1: The HA ester may be shaped (step 17) and crosslinked(step 31) then further processed (pathway 32 to option 38);saponification/hydrolysis may be performed on the HA ester (pathway 33 ato step 34); or HA ester may simply be used/further processed (33 a tooption 38) prior to being returned to a regenerated HA structure. Thethermally formed structure(s)/piece(s)/etc. may be used ‘as is’ andfurther processed as necessary (box 39 a) or further processed and builtinto an assembly/system (e.g., may be integrated into a conventionalstructure), sterilized, and so on (box 39 b)—depending upon finalapplication of the end-product. For example, regenerated HA may behot-molded with thermoplastic ultra high molecular weight polyethylenefor joint replacements implants, or the HA esters can be molded intoanimal tissue scaffold.

As mentioned, the applicants hereof have published earlier work: USPatent App. US 2003/0083433 A1 filed on behalf of the assignee hereoffor the applicants. In this earlier work, applicants provide backgroundas to silylation of HA complexes.

Begin Quoted Text:

Example 2

-   -   By way of further examples the following is offered: In the case        of using a guest of HA, which is strongly hydrophilic with its        many polar groups (—COOH, —OH and —CONHCH₃) on its long        molecular chain, diffusion of HA molecules directly into a bulk        UHMWPE structure is difficult. Therefore a modification of the        HA molecules is done to increase hydrophobicity and        compatibility with both UHMWPE and organic solvents used in        connection with cloaking.    -   2A) Silylation of HA to increase its hydrophobicity: Silylation        is a known technique for increasing hydrophobicity, and        createorganic-soluble derivatives of substances. During a        silylation reaction of HA, the hydrophilic groups containing        active hydrogen, such as —COOH, —OH, and —NH₂, are masked by        hydrophilic silyl groups. The reaction is reversible, the        silylated functional groups can be returned to their original        state through hydrolysis reaction. HA is a muco-polysaccharide        of molecular weight up to millions (˜10⁶). Compared with        silylation of poly-L-lysine (MW=˜1000), silylating HA is        difficult due to its large molecular weight. In contrast to PLL        silylation previously performed by the applicants (see above),        preferably HA is modified before silylation to increase its        solubility in silylation solvents (polar organic solvents can be        used). The steps include:        -   (1) Reaction of HA with long-chain aliphatic quaternary            ammonium salts (QN⁺). Polyanions, such as HA, combined with            certain organic cations, such as paraffin chain ammonium            (QN⁺) ions, produces a precipitable complex. The complex is            a true salt of the polyacid and quaternary base. HA was            modified with long-chain aliphatic ammonium salts, to            improve its solubility in organic solvents. Combination of            QN+ with polyannions occurs in those pH ranges in which the            polyannions are negatively charged. The reaction between HA            and ammonium cations in water can be expressed:            HA⁻-Na⁺+QN⁺A⁻→HA⁻-QN⁺↓+Na⁺A⁻        -    where HA-Na⁺ is the sodium salt of hyaluronic acid; HA⁻-QN⁺            is the precipitable complex between HA carboxylic polyanion            and long chain paraffin ammonium cations.            HA⁻-QN⁺(HA-CPC/HA-CTAB) complexes were used. The complexes            (HA⁻-QN⁺) precipitated from HA aqueous solution are soluble            in concentrated salt solutions, so HA can be recovered from            its insoluble complexes. Ammonium salts used were:            cetyltrimethylammonium bromide monohydrate (MW: 358.01)            (CTAB) and cetylpyridinum chloride (M.W. 364.46) (CPC).        -   (2) Silylation of HA⁻-QN⁺ complexes: HA-CPC and HA-CTAB were            silylated in DMSO solution with BSA, HMDS and other typical            silylation agents. Silylation agents are generally sensitive            to humidity, silylating operation should be under the purge            of dry N₂.    -   2B) Acylation of HA to improve its thermal flow: To make HA        flowable at high temperature, the strong hydrogen bonding        between its molecules must be disrupted, and the molecular order        (i.e., crystallinity) of HA needs to be destroyed. Acylating the        hydroxyl groups on HA with long-chain aliphatic carboxylic acids        chloride will help in de-crystallizing HA. Acid chlorides, from        caproyl to stearoyl chloride, can be used as acylating agents.        Acylation is a known process for disrupting crystallinity in        other polysaccharides. Acylation reactions are performed in        solution (of HA⁻-QN⁺ in DMSO, for example). Start with a DMSO        solution of HA⁻-QN⁺ complex using the technique described above        in connection with Silylation of HA, above. Acylation is done to        make the hydrophilic guest hydrophobic enough to be molded with        a hydrophobic host, such as UHMWPE, without phase separation.    -   2C) Entanglement by Swelling of Host to Facilitate Diffusion of        Guest: . . .    -   2D) Entanglement by using Porous (eg. UHMWPE) Host Structure: .        . .    -   2E) Entanglement by Powdered Mixture which can be Molded: . . .

End Quoted Text.

Once again, in connection with reviewing the example(s) depictedthroughout, one will appreciate that a variety of alternative acidchlorides are suitable for use as identified in the results reported inTABLE 1. Results of the solubility of HA esters as investigated, areidentified for reference, in TABLE 2.

TABLE 1 Properties of HA and its aliphatic esters Starting Melting Pointof Acid Formula Point Degradation Material Chlorides of Acid Chlorides(° C.) (° C.) HA No* 212 HA-CTA No* 175 Silyl HA-CTA No* 161 HA CaproateHexanoyl CH₃(CH₂)₄COCl 191.7 170 HA Caprylate Octanoyl CH₃(CH₂)₆COCl188.9 184 HA Caprinate Decanoyl CH₃(CH₂)₈COCl 156.7 187 HA LaurateLauroyl CH₃(CH₂)₁₀COCl 97.8 191 HA Palmitate Palmitoyl CH₃(CH₂)₁₄COCl96.4 200 HA Stearate Stearoyl CH₃(CH₂)₁₆COCl 88.3 194 *No meltingtemperature: polymer degrades before melting.

TABLE 2 Solubility of HA esters in organic solvents HA Esters DMSO THFAcetone Xylenes Hexane HA Caproate swollen + + + − HA Caprylateswollen + + + − HA Caprinate swollen + + + − HA Laurate − + + + − HAPalmitate − − − + + HA Stearate − − − + + “+” soluble; “−” insoluble.

1.0 EXAMPLE

A series of melt-processable hyaluronan (HA) esters were synthesized. Asilylated complex of HA with cetyltrimethylammonium cations (silylHA-CTA) was used as the starting material. Reactions were performed withacid chlorides as the acylation agents in both xylenes or no solventother than the acid chloride. The disappearance of all characteristicFT-IR vibration bands associated with the —OSi(CH₃)₃ groups and theappearance of the strong ester carbonyl peak at 1753 cm⁻¹ demonstratedsuccess of esterification (step 16, FIG. 1). Thermoplasticity wasachieved when length of aliphatic chains in the HA ester was equal to orgreater than 10 carbon atoms: The longer the ester chain, the lower themelting point (TABLE 1). As uniquely identified herein, to meet targetedspecifications—different melting temperatures—an adjustment can be madeto the acid chloride chain length.

It is known that long paraffin-chain quaternary ammonium compounds,including cetyltrimethylammonium bromide (CTAB) and cetylpyridiniumchloride (CPC), can be used to precipitate polyanions of hyaluronan andvarious sulfuric polysaccharides from aqueous solution aspolysaccharide-ammonium salt complexes. These prior complexes wereactually salts between polysaccharide acids and quaternary ammoniumbases, which were soluble in salt solutions, such as sodium chloride ofvarying concentrations. For example, HA-CP complex is soluble at sodiumconcentrations above 0.2 N, while the complexes with sulfatedpolysaccharides (such as keratosulfate, chondroitin sulfate, heparin andso on) usually require higher salt concentrations for solubility. Here,to synthesize HA esters from HA-aliphatic quaternary ammonium saltcomplexes: The dissociated carboxyl group on hyaluronan (HA) is combinedwith aliphatic quaternary ammonium cations for separation from otherpolysaccharides and as an intermediate for further modificationaccording to the invention.

1.1 Materials. Silyl HA-CTA was obtained using sodium hyaluronate(HyluMed®, medical grade, MW: 1.36×10⁶ daltons) from Genzyme (Cambridge,Mass.). Acid chlorides, including hexanoyl (caproyl), octanoyl(capryloyl), decanoyl (caprinoyl), lauroyl, palmitoyl, and stearoylchlorides (TABLE 1), are available from Aldrich (Milwaukee, Wis.).Xylenes, hexane, acetone, dimethylsulfoxide (DMSO), tetrahydrofuran(THF), pyridine, and potassium hydroxide are available from Fisher(Pittsburgh, Pa.). Ethanol (ACSIUSP grade) is available from Pharmco(CT).

1.2 Synthesis of HA esters. Silyl HA-CTA, 200 mg, was added to 5-10equivalents of acid chlorides under N₂ atmosphere. The mixture washeated for 0.5-1 h at 80° C. Once cooled, the reaction product solutionbecame a turbid, viscous paste or, in the case of HA stearate, a solid.Hexane was used to precipitate the HA caproate, caprylate, caprinate andlaurate from the product mixtures. Acetone was used to wash the excessof palmitoyl and stearoyl chlorides from the HA esters. Afterprecipitating or washing, the resulting HA esters were dried in a vacuumoven until constant weight was obtained. When performing synthesis withsolvents, xylenes were used to form a homogeneous solution and promotereaction. After esterification, xylenes were removed as well as excessacid chlorides were also removed.

1.3 Saponification of HA esters. For those esters soluble in acetone,such as HA caproate, caprylate, caprinate and laurate, 200 mg of HAester were dissolved in 20 ml of acetone-ethanol mixture solvent (v/v1:1), forming a clear solution. Aqueous 1M KOH solution (5 ml) wasslowly added to the solution. The saponified HA ester graduallyprecipitated from solution. After addition of 5 ml water, the solutionstood at room temperature for another hour to saponify the esterresidue. The precipitate was filtered and the KOH was removed bydissolving the precipitate in water and re-precipitating with ethanol.The final white particle product, regenerated HA, was vacuumed drieduntil a constant weight was obtained. For those HA esters insoluble inacetone, such as HA palmitate and stearate, 200 mg of ester wasdissolved in a pyridine-ethanol mixture solvent.

1.4 Fourier Transform Infrared Spectroscopy (FT-IR). A Nicolet Magna-IR760 Spectrometer (E.S.P.) was used to record FT-IR spectra. Transmissionabsorption spectra were collected over a range 600-4000 cm⁻¹ at aresolution of 4 cm⁻¹ with 128 scans. HA caproate, caprylate, caprinateand laurate were dissolved in acetone and coated on NaCl disks for FT-IRanalysis. HA palmitate and stearate were dissolved in xylenes and castonto NaCl disks. Regenerated HA from ester saponification was mixed withKBr and pressed into pellets.

1.5 X-Ray Photoelectron Spectroscopy (XPS). XPS analyses were performedon a PHI 5800 spectrometer (Physical Electronics, Inc., MN).Measurements were taken with an electron takeoff angle of 45° from thesurface normal (sampling depth ˜50 Å). High-resolution spectra (C1s,N1s) were obtained at a pass energy of 25 eV. Component peak analysis ofhigh-resolution spectra was performed using XPSPeak 4.1 software. HAlaurate was dissolved in xylenes and cast into a film on a glass slidefor XPS analysis.

1.6 Differential Scanning Calorimetry (DSC) and Thermal GravimetricAnalysis (TGA). The thermal properties of the HA esters were determinedusing Seiko DSC SCC 2200 differential scanning calorimeter and Seiko TGSCC 5200 thermal gravimetric analysis at a heating rate of 10 C/min inair.

1.7 Synthesis of HA Esters. Compared with native HA, silyl HA-CTA isuseful as an intermediate of further modifications of HA, includingesterification. The trimethylsilyl groups of silyl HA-CTA are moreeasily replaced than the active hydrogen in HA hydroxyl groups.Esterification with silyl HA-CTA as a starting material (FIG. 1) wascarried out without addition of catalysts, and the reaction took placeat a high rate (within 1 hr). The by-product trimethylchlorosilane(TMCS), which has a low boiling point (57° C.), can be evaporated at thereaction temperature (80° C.). Acylation takes place at the oxygen ofthe trimethylsilyloxy group —O—Si(CH₃)₃ in the silyl HA-CTA (FIG. 2B).The FT-IR spectrum of HA palmitate is shown in FIG. 3 in comparison withHA-CTA (complex between HA and with cetyltrimethyl ammonium salt) andsilyl HA-CTA. The spectra of all HA esters are similar, so the spectrumof HA palmitate is used as a representative. Results indicate theintroduction of a large amount of acyl groups with acylation.

The C1s XPS high-resolution spectrum of HA laurate is shown in FIG. 4A.In comparison with silyl HA-CTA (FIG. 4B), the C3 component of the HAester increases significantly, and its intensity almost doubles that ofthe C2 peak. In silyl HA-CTA, the C3 peak area is inferior to C2. Theincrease of C3 peak percentage and intensity relative to C2 in HAlaurate can be explained with the introduction of large amounts of estergroups during esterification.

The N1s XPS high-resolution spectra of HA laurate and silyl HA-CTA areshown respectively in FIGS. 5A, 5B. The N1s signal of the ester consistsof two components: ammonium salt N⁺ (402.8 eV) and amide N (399.8 eV).The intensity of ammonium salt N⁺ signal is smaller than that of amide Nsignal in the ester sample, but they are almost equal to that observedin silyl HA-CTA. While this might suggest that the acid chloride alsoattacked -CTA groups during esterification, any such attack was notdominant, because most ammonium salt N⁺ groups still remain.

1.8 Regeneration of HA from HA Esters. The FTIR spectrum of HAregenerated from saponification of HA caprylate is shown in FIG. 6. Thepeaks characteristic of absorptions for -CTA and capryloyl esters aregone. There is no difference between the spectra of regenerated HA andoriginal HA, indicating the substantial removal of CTA and ester groupsthrough saponification. Alkaline metal cations (e.g. Na⁺ and K⁺) withsufficiently high concentration can displace the CTA groups in HA ester,resulting in a regenerated HA (FIG. 6). A mixture of acetone and ethanolwas used for saponification of the HA esters soluble in acetone,including HA esters from caproate to laurate. HA palmitate and stearateare insoluble in acetone, but dissolve in pyridine, so apyridine-ethanol mixture was used for them.

1.9 Properties of HA Esters. From the TGA data in FIG. 7, it can be seenthat HA begins to degrade around 212° C., but no melting point can befound below that temperature in the DSC data (FIG. 7). Each disaccharideunit of HA contains four hydroxyl groups, one amide and one carboxylgroup. Due to the strong intra- and inter-molecular hydrogen bonds, HAis highly crystalline and insoluble in organic solvents, and cannot betransformed into the molten state before its decomposition.

Introduction of aliphatic acyl groups to HA disrupted the strong HAintermolecular bonding, reducing the crystallinity and producingappreciable thermoplasticization. For HA laurate, a broad endotherm isobserved ranging from 76° C. to 105° C. with a peak at 97.8° C., whileHA caproate shows a melting peak starting from 175° C. with the peak at191.7° C. The TGA results (FIG. 7) show that HA laurate begins degradingat about 191° C., while HA caproate begins degrading at approximately170° C.

The melting temperature and the starting points of degradation forvarious HA esters are summarized in TABLE 1. The intermolecularinteraction between the polymer chains decreases with increasing lengthof ester side chains, as indicated by the change in melting points. Thehigher aliphatic acid chloride more effectively conferredthermoplasticity to HA. Starting from HA caprinate, higher aliphaticesters of HA melt far before the degradation begins, and thus can bemelt-processed. Note that, for caproyl and capryloyl: HA caproate andcaprylate did not achieve thermal fluidity before the onset ofdegradation. Degradation points of HA esters depend on the balancebetween two factors: a decrease in crystallinity (molecular order) andan increase in the length of acyl groups. From HA caproate to palmitate,the effect of acyl group chain length seems to dominate. However, withHA stearate, the effect of molecular order disruption seems to dominate.Thus, esterification with high aliphatic acid chlorides is an effectivemethod for imparting thermoplasticity to HA. HA caprinate, laurate,palmitate and stearate had melting peaks far below their degradationtemperatures, providing sizable ‘safe-harbor’ for thermalmelt-processing of the polymers. To the extent acyl groups are not longenough to disrupt the strong HA intermolecular interactions andmolecular arrangement (e.g., caproyl and capryloyl), their correspondingHA esters are not hot melt-processable.

The solubility of HA esters were also investigated and the results aresummarized in TABLE 2. HA esters from caproate to laurate are soluble inacetone and THF, while palmitate and stearate are soluble in hexane,indicating that the hydrophobicity of HA esters increases with the sidechain length. DMSO may not be a good solvent for the HA esters, whilexylenes are a good solvent for these esters.

The resultant thermoplastic HA esters can be hot molded into films,sheets, and any desired shapes. They can be used alone or mixed withsome other biomedical grade thermoplastics, such as ultra high molecularweight polyethylene. Crosslinkers, such as blocked polyisocyanate, canalso be used during molding to obtain a permanent three-dimensional HAester network (see, also, below discussion). Finally, the acyl groupscan be easily removed through saponification in order to return HA to aregenerated state after molding.

2.0 Crosslinking Background: Crosslinking of regenerated HA and uniqueHA derivatives synthesized according to the invention (see FIG. 1,optional steps identified at 31 and 36 by way of example).

Several different methods are contemplated hereby to crosslinkhyaluronan. By controlling the extent of crosslinking, the type ofcovalent bond and the hyaluronan group involved, it is possible tocreate a wide range of physically diverse materials from highlyviscoelastic solutions to insoluble gels or solids.

2.1 Crosslinking through Hydroxyl Groups.

HYLAN, as referenced above, is a hyaluronan derivative(s) with thehydroxyl groups crosslinked, while leaving the carboxyl and acetamidogroups unreacted. Retention of carboxyl groups was important, here,because the polyanionic character of hyaluronan plays a lead role in itsphysicochemical and biological properties, Balazs et al. HYLAN polymerscurrently used in medicine are HYLAN A and HYLAN B. HYLAN A is aviscoelastic fluid, developed by Balazs et al. by cross-linkinghyaluronan chains to specific protein molecules with formaldehyde duringrecovery of the polymer from animal tissues.

HYLAN B is synthesized by reacting hyaluronan with divinyl sulphone inaqueous alkaline solutions at room temperature. With this reagentbis-(ethyl) sulphone crosslinks are formed, producing an infinitehyaluronan network that is no longer water soluble. The degree ofcrosslinking can be controlled to create a wide range of materials thatrange from soft deformable gels to solid membranes and tubes, withprolonged or permanent residence times.

Polyfunctional epoxy compounds may be used to crosslink hyaluronan inalkali solution with a water-soluble organic solvent present, such asacetone and methanol, which may prevent the HA from decomposing.Depending on the ratio of epoxy compound to HA, different crosslinkedpolymers can be obtained. Products with a small molar ratio (<10) aresoluble, while those with a high ratio (>10) are insoluble.

A similar crosslinked insoluble HA polymer has been developed byTomihata et al. (1997) through the reaction of poly(ethylene glycol)diglycidyl ether (a diepoxy compound) with HA under acidic or neutralconditions. This crosslinked HA film has good degradation-resistance.

Phosphate-crosslinked HA is a type of ester obtained by esterifying HAwith phosphoric acid derivatives, such as phosphoryl chloride (POCl₃),in an alkaline medium. This reaction occurs very quickly with gelformation within a few minutes, and the excess of crosslinking agent iseasily hydrolyzed and removed, Malson, et al., (1998).

A high degree of HA crosslinking has been achieved by reactingglutaraldehyde (GA) with an HA film in an acetone-water mixture underhydrochloric acid, Tomihata et al., (1997). The intermolecular formationof hemiacetal bonds between GA and the hydroxyl groups of HA led tocrosslinking. The crosslinked product tends to be stable in phosphatebuffer solution.

Polyisocyanates were used to permanently crosslink HA in anhydrousorganic solvents, Balazs et al., (1987), such as acetone, or toimmobilize HA coatings on polymeric surfaces. Due to the stability ofthe urethane linkage, the crosslinked products are durable and can beresistant to decomposition in aqueous solutions. Blocked isocyanatesthat do not become active until a certain temperature is reached may beused to crosslink and mold the unique HA ester of the invention in asingle step such that crosslinking occurs after flow and consolidation.

2.2 Crosslinking through Carboxyl Groups.

In the crosslinking reactions identified above in section 2.1, HA playsthe role of a polymeric poly-hydroxyl component. Another category ofcrosslinked HA derivatives can be obtained through reactions in which HAplays the role of a poly-carboxylic acid.

Ionic crosslinking reactions are possible through the reaction of HAcarboxyl groups and polyvalent cations. BaCl₂, CaCl₂ and FeCl₃ were usedby Halpern et al. (1989) to treat HA coatings, producing stableproducts. INTERGEL®, an anti-adhesion product of Lifecore, is a hydrogelof HA formed by chelation with ferric hydroxide.

A polymeric network of HA can also be synthesized via three- orfour-component condensation known, respectively, as the Passerinireaction and the Ugi reaction, Crescenzi et al., (1998 & 2003). In thePasserini reaction, a given amount of water-soluble dialdehyde (e.g.glutaraldehyde) and a highly reactive isocyanide (e.g.cyclohexylisocyanide) were added to HA solution with pH 3.5-4.0, whichis stirred at room temperature until a gel forms. In Ugi four-componentcondensation, the mixture contains HA, formaldehyde,cyclohexylisocyanide and lysine ethyl ester. The degree of crosslinkingis controlled by the amount of dialdehyde in the Passerini reaction anddiamine in the Ugi reaction. Hydrogels obtained from both reactions aretransparent and mechanically stable.

2.3 Crosslinking through Both Hydroxyl and Carboxyl Groups.

ACP™ is an auto-crosslinked HA product developed by Fidia AdvancedBiopolymer used for adhesion prevention or tissue engineering scaffolds.ACP™ is generated by condensation between the hydroxyl and carboxylgroups of hyaluronan.

A low-water content hyaluronan hydrogel film was made by crosslinking anHA film in an aqueous mixture containing an organic solvent (ethanol oracetone) and a water-soluble carbodiimide (WSC). WSC does not chemicallybind to HA molecules, but it mediates the reaction. Ester bonds formedbetween hydroxyl and carboxyl groups on different HA molecules result incrosslinking.

While certain representative embodiments, examples, and details havebeen shown merely for the purpose of illustrating the unique HA materialand associated method of producing, including any program code utilizedto instruct automated manufacturing/fabrication machinery employed tocarry out process of producing according to the invention, those skilledin the art will readily appreciate that various modifications, whetherspecifically or expressly identified herein, may be made to any of therepresentative embodiments without departing from the novel teachings orscope of this technical disclosure. Accordingly, all such modificationsare contemplated and intended to be included within the scope of theclaims. Although the commonly employed preamble phrase “comprising thesteps of” may be used herein in a method claim, applicants do not intendto invoke 35 U.S.C. §112 ¶6. Furthermore, in any claim that is filedherewith or hereafter, any means-plus-function clauses used, or laterfound to be present, are intended to cover at least all structure(s)described herein as performing the recited function and not onlystructural equivalents but also equivalent structures.

1. A process for producing a hyaluronan (HA) ester, the processcomprising the steps of: (a) performing a silylation reaction on anHA-quaternary ammonium complex; and (b) performing an acylation reactionon the silyl HA-quaternary ammonium complex using an acid chloride. 2.The process of claim 1 wherein: (a) the step of performing a silylationreaction comprises silyating an HA-cetyltrimethyl ammonium salt complex,HA-CTA, producing a silyl HA-cetyltrimethyl ammonium salt complex; and(b) the step of performing an acylation reaction comprises introducingthe acid chloride having been selected from aliphatic acyl groupsconsisting of: Hexanoyl, CH₃(CH₂)₄COCl; Octanoyl, CH₃(CH₂)₆COCl;Decanoyl, CH₃(CH₂)₈COCl; Lauroyl, CH₃(CH₂)₁₀COCl; Palmitoyl,CH₃(CH₂)₁₄COCl; and Stearoyl, CH₃(CH₂)₁₆COCl.
 3. The process of claim 2further comprising the step of shaping the hyaluronan(HA) ester byapplying thermal energy to melt-process the ester into astructure-shape.
 4. The process of claim 2 further comprising the stepsof: (a) shaping the hyaluronan (HA) ester into a structure-shape; and(b) performing a saponification substantially removing acyl groups,—CH₃(CH₂)_(N)CO, and the cetyltrimethyl ammonium salt groups, -CTA, fromthe hyaluronan (HA) ester to produce a regenerated HA.
 5. The process ofclaim 1: (a) wherein the step of performing a silylation reactioncomprises silyating an HA-cetyltrimethyl ammonium salt complex, HA-CTA,producing a silyl HA-cetyltrimethyl ammonium salt complex; and (b)further comprising the step of performing a saponification substantiallyremoving acyl groups and the cetyltrimethyl ammonium salt groups, fromthe hyaluronan (HA) ester to produce a regenerated HA.
 6. The process ofclaim 1: (a) wherein the step of performing an acylation reactioncomprises introducing the acid chloride having been selected fromaliphatic acyl groups consisting of: Hexanoyl, CH₃(CH₂)₄COCl; Octanoyl,CH₃(CH₂)₆COCl; Decanoyl, CH₃(CH₂)₈COCl; Lauroyl, CH₃(CH₂)₁₀COCl;Palmitoyl, CH₃(CH₂)₁₄COCl; and Stearoyl, CH₃(CH₂)₁₆COCl; and (b) furthercomprising the step of shaping the hyaluronan (HA) ester into astructure-shape while crosslinking the hyaluronan (HA) ester.
 7. Aprocess for producing a hyaluronan (HA) ester, the process comprisingthe steps of: (a) performing a silylation reaction on anHA-cetyltrimethyl ammonium salt complex, HA-CTA; and (b) performing anacylation reaction on the silyl HA-cetyltrimethyl ammonium salt complexusing an acid chloride selected from the group consisting of: Hexanoyl,CH3(CH₂)₄COCl; Octanoyl, CH₃(CH₂)₆COCl; Decanoyl, CH₃(CH₂)₈COCl;Lauroyl, CH₃(CH₂)₁₀COCl; Palmitoyl, CH₃(CH₂)₁₄COCl; and Stearoyl,CH₃(CH₂)₁₆COCl.
 8. The process of claim 7 further comprising the stepof: (a) shaping the hyaluronan (HA) ester into a structure-shapeselected from the group consisting of: a plurality of polymer fibers; agenerally solid bulk structure; and porous bulk structure; and (b)performing a saponification substantially removing acyl groups and thecetyltrimethyl ammonium salt groups, from the hyaluronan (HA) ester toproduce a regenerated HA.